Allstars hs cell death control

In the large scale screen, each target gene was knocked down with a pool of four unique siRNAs. Cell lines representing human triple negative (HCC70) and hormone receptor negative HER2 positive breast cancer subtype (SKBR3) were compared to a non-malignant human breast cell line (MCF10F). All cell lines were purchased from ATCC (Manassas VA) and tested for mycoplasma by the universal mycoplasma detection kit (ATCC) prior to the large scale screens. Transfection conditions, cell number, timing, and reagent concentration were determined by feasibility studies that identified optimal assay conditions to obtain a ~20-fold window using a positive apoptosis control siRNA associated with cell survival (Kif11 siRNA, Qiagen Germantown PA), and universal negative control siRNA (AllStars Negative Control siRNA, Qiagen). For confirmation of the large scale screen, the pooled siRNA was deconvoluted, manually transfected in 96-well format, and assayed for cell survival and apoptosis. The positive control was AllStars HS Cell Death Control (Qiagen). For the large scale screens, candidate antigens were chosen based on (a) ≥30% decreased viability and (b) ≥1.5-fold increased apoptosis in the invasive breast cancer cell lines, but not in the non-malignant cell line.

Cells were seeded in six-well culture plates in complete medium at 2 × 10⁵ cells/mL shortly before transfection and incubated under standard conditions until transfection. The cells were transfected with 10 nM scrambled siRNA (AllStars Negative Control siRNA, Qiagen) or 10 nM siRNA targeted against both main isoforms of PTPN2 (siRNA_15 Qiagen) using HiPerFect reagent (Qiagen), following the manufacturer’s fast forward protocol. After 48 h, cells were used for follow-up experiments. A positive control siRNA (AllStars Hs Cell Death Control, Qiagen), knocking down ubiquitous human cell survival genes, was included in every experiment. All positive controls performed resulted in > 90% cell death as visualised by light microscopy. Unspecific effects of transfection reagents were discarded by mock-transfecting the cells during the optimisation of the experiments. Knockdown control was performed using Western blot and gene expression analysis (at least 80% knockdown per experiment; results not shown).